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. 2016 Jan 27;36(4):1071–1085. doi: 10.1523/JNEUROSCI.2430-15.2016

Figure 6.

Figure 6.

Microtubule orientation in neurons before and after polarization. A, Schematic representation of the microtubule LS procedure in a neurite. P indicates the proximal region and D the distal region. B, A representative DIV1 cortical neuron expressing GFP–MT+TIP, followed by the maximum projection and stills from a time-lapse recording of the indicated neurite. The dashed cyan line represents the region of LS, and the cyan arrow indicates the moment of laser severing. Green and red arrows mark selected plus- and minus-end-out microtubules, respectively. C–E, Kymographs and illustration of microtubule tracings from representative time-lapse recordings of a neurite (C) from a nonpolarized neuron, dendrite (D), and axon (E) of a polarized neuron. Green lines represent plus-end-out microtubules and red lines minus-end-out microtubules. Cyan asterisk and arrow indicate time and location of LS. F, G, Quantification of anterograde (F) and retrograde (G) growing GFP–MT+TIP comets, with and without LS (n = 10–18 neurons) in DIV1 cortical neurons. H, Percentage of microtubules with their minus ends out in unpolarized and polarized DIV1 neurons, before (black columns) and after (gray columns) LS; n = 10–18 neurons. I, Representative images of nonpolarized (top) and polarized (bottom) DIV1 hippocampal neurons stained for both CAMSAP2 (left; I, L) and α-tubulin (right). J, Representative images of stage 2 neurons transfected with GFP and control pSuper (top) or CAMSAP2 shRNA (bottom) and stained for both GFP (left) and CAMSAP2 (right). K, Quantification of CAMSAP2 intensity in neurites in control and CAMSAP2 shRNA transfected neurons (n = 19 neurites). ***p < 0.001 using t test. L, Quantification of CAMSAP2 localization in hippocampal neurons before and after polarization (n > 100 neurites). CAMSAP2 distribution within the neurites is classified in indicated categories. M–O, Representative examples of endogenous CAMSAP2 intensity profiles in neurites (M), before polarization, dendrites (N), and axons (O) from DIV1 hippocampal neurons. Scale bars: B, 2 and 10 μm; C–E, I, J, 5 μm; I, J, 20 μm. Error bars indicate SEM.